Identification of host genetic factors modulating β-lactam resistance in <i>Escherichia coli</i> harbouring plasmid-borne β-lactamase through transposon-sequencing
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Since β-lactam antibiotics are widely used, emergence of bacteria with resistance to them poses a significant threat to society. In particular, acquisition of genes encoding β-lactamase, an enzyme that degrades β-lactam antibiotics, has been a major contributing factor in the emergence of bacteria that are resistant to β-lactam antibiotics. However, relatively few genetic targets for killing these resistant bacteria have been identified to date. Here, we used a systematic approach called transposon-sequencing (Tn-Seq), to screen the <i>Escherichia coli</i> genome for host genetic factors that, when mutated, affect resistance to ampicillin, one of the β-lactam antibiotics, in a strain carrying a plasmid that encodes β-lactamase. This approach enabled not just the isolation of genes previously known to affect β-lactam resistance, but the additional loci <i>skp</i>, <i>gshA</i>, <i>phoPQ</i> and <i>ypfN</i>. Individual mutations in these genes modestly but consistently affected antibiotic resistance. We have identified that these genes are not only implicated in β-lactam resistance by itself but also play a crucial role in conditions associated with the expression of β-lactamase. <i>GshA</i> and <i>phoPQ</i> appear to contribute to β-lactam resistance by regulating membrane integrity. Notably, the overexpression of the uncharacterized membrane-associated protein, <i>ypfN</i>, has been shown to significantly enhance β-lactam resistance. We applied the genes identified from the screening into <i>Salmonella</i> Typhimurium and <i>Pseudomonas aeruginosa</i> strains, both critical human pathogens with antibiotic resistance, and observed their significant impact on β-lactam resistance. Therefore, these genes can potentially be utilized as therapeutic targets to control the survival of β-lactamase-producing bacteria.
由于β-内酰胺类抗生素（β-lactam antibiotics）应用广泛，对其产生耐药性的细菌的出现已对社会构成严重威胁。具体而言，编码β-内酰胺酶（β-lactamase，一种可降解β-内酰胺类抗生素的酶）的基因的获取，是β-内酰胺类耐药菌出现的主要诱因之一。然而，截至目前，可用于杀灭这类耐药菌的遗传靶点仍相对较少。本研究采用转座子测序（transposon-sequencing, Tn-Seq）这一系统性方法，在携带编码β-内酰胺酶质粒的大肠杆菌（Escherichia coli）菌株中，筛选宿主遗传因素中发生突变后会影响氨苄青霉素（ampicillin，一种β-内酰胺类抗生素）耐药性的基因。该方法不仅成功分离出此前已被报道与β-内酰胺类耐药性相关的基因，还额外鉴定出了skp、gshA、phoPQ以及ypfN这几个基因座。这些基因的单个突变虽程度温和，但始终会对抗生素耐药性产生影响。本研究发现，这些基因不仅自身参与β-内酰胺类耐药性的调控，还在与β-内酰胺酶表达相关的生理过程中发挥关键作用。其中gshA与phoPQ似乎通过调控细胞膜完整性来增强β-内酰胺类耐药性。值得注意的是，未表征膜相关蛋白ypfN的过表达已被证实可显著提升β-内酰胺类耐药性。本研究将筛选得到的这些基因应用于鼠伤寒沙门氏菌（Salmonella Typhimurium）与铜绿假单胞菌（Pseudomonas aeruginosa）这两种具有抗生素耐药性的重要人类病原菌菌株中，观察到它们对β-内酰胺类耐药性具有显著影响。因此，这些基因有望作为治疗靶点，用于调控产β-内酰胺酶耐药菌的存活。
